Clostridium difficile is an obligatory anaerobic, Gram-positive rod bacterium which is 3-6 μm long and approximately 0.5 μm wide. The Clostridium genus belongs to the Bacillaceae family. The discovery of C. difficile as an important cause of antibiotic-associated colitis occurred at the end of the 1970s. The name C. difficile is a reference to the slow growth in culture and difficulty with isolating the bacterium. On supplemented blood agar dishes, large translucent grey colonies grow without haemolysis. In common with all Clostridia, C. difficile can produce spores. In this manner, the bacteria can also survive under extreme environmental conditions.
Healthy adults have 2-7% C. difficile in their gastrointestinal tract. During antibiotic therapy, the protective effect of the physiological flora is perturbed and an overgrowth of C. difficile may occur in the gut. The resulting clostridium difficile associated disease (CDAD) varies in severity as a function of the virulence of the C. difficile strain which is present. Clinical presentation varies from mild diarrohea to severe pseudomembranous colitis with fever and cramp-like stomach pain and the risk of severe complications such as colon perforation, sepsis and toxic megacolon. The primary virulence factors are an enterotoxin (toxin A) and a cytotoxin (toxin B). The toxins belong to the large clostridial toxin (LCT) family and have glycosyltransferase activity. Furthermore, some strains carry the genes for the binary CDT toxin (ADP-ribosyltransferase) which is considered to be a further virulence factor.
C. difficile strains can be divided into so-called standard strains and their variants. Strains with toxinotype 0 are generally described as standard C. difficile strains. These include strains with different ribotypes, for example 001, 003 and 012. Strains with toxinotype 0 are characterized by the presence of the genes for both toxins A and B, however they lack the genes for the binary toxin.
In recent years, it has been shown that in addition to standard C. difficile strains, there also exist a plurality of different variant C. difficile strains which are of growing clinical importance and which have also been detected in house pets (in particular dogs and cats) and livestock (in particular calves and pigs).
In this regard, endemic, frequently severe infections with variant C. difficile strains have been observed since 2003 in Canada, the USA, Great Britain and many other European states.
Isolated variant C. difficile strains associated with these severe infections have both the genes for toxin A (tcdA) and B (tcdB) as well as the genes for the CDT and have partial deletions in their regulatory gene tcdC.
These highly virulent C. difficile strains from PCR-ribotype 027, toxinotype III, REA-B1 and PFGE NAP1 have an 18 by deletion in the regulatory gene tcdC and a frameshift in position 117, presumed to be the cause of its increased toxin production in vitro and a reason for its increased virulence. These strains are also characterized in that in comparison with other C. difficile strains, they have altered surface proteins and increased adhesion to human gut epithelial cells. In addition, these strains are resistant to antibiotics of the fluoroquinolone class and to the antibiotic erythromycin—presumably because of mutations in the genes for gyrA/B and 23sRNA.
These highly virulent C. difficile strains will hereinafter be referred to by the abbreviation “C.d.-027-hv”.
While earlier, an infection with C. difficile affected mainly patients over 60 years of age, C.d.-027-hv has also been detected in younger patients and also outside hospitals in patients deemed until now to be low risk. Compared with previously detected C. difficile strains, it is five times more lethal.
Another group of clinically relevant variant C. difficile strains is classified as ribotype 017, toxinotype VIII and serotype F. These strains are essentially characterized in that they have a 1.7 kb deletion in the tcdA gene and because of an early stop codon, they do not express toxin A. The strains have antibiotic resistance to clindamycin and fluoroquinolone, they lack the gene for the binary CDT toxin, and in addition the regulatory gene tcdC has no deletions. Increasing incidences of such strains have been reported in the USA, Japan, Israel, Ireland and the Netherlands, inter alia. The mortality in patients with such infections, at 14-66%, is higher than that for the standard strains.
These highly virulent C. difficile strains will hereinafter be referred to by the abbreviation “C.d.-017-hv”.
Diagnostic typing of C. difficile is usually carried out by detecting toxin A, and in some cases by the combined detection of toxin A and toxin B. Variant C. difficile strains cannot be detected using this method. The variant strains are either not detected at all (toxin A negative strains) or not detected separately.
In order to identify and characterize variant C. difficile strains, they have to be typed. Methods for typing C. difficile, for example toxinotyping, serotyping, ribotyping or typing using PFGE or REA, are known in the art. All of those methods, however, are work and time intensive, can only be carried out by specialized laboratories and also require complex prior isolation of the C. difficile strains in question.
In order to identify specific C. difficile strains precisely, moreover, further investigations are usually required, such as in the case of C.d.-027-hv the detection of antibiotic resistance and of the deletion in the tcdC gene.
Therefore, in the laboratory, when for example investigating the presence of a C.d.-027-hv, the following is carried out:                1. General detection of C. difficile by detecting toxin A and/or B, in the stool of the patient using immunoassay;        2. if positive: culture of C. difficile bacteria from the material being investigated;        3. following successful isolation of bacteria: test for presence of erythromycin and moxifloxacin resistance in E-test;        4. following positive detection of resistance: test for presence of ribotype 027 using a DNA preparation and PCR amplification of ribosomal RNA gene (ribotyping) [FIG. 1];        5. following positive detection of ribotype 027: partial genotyping to detect the two toxin genes tcdA and TcdB, the cdtA/B gene for the binary CDT toxin and the deletions in the regulatory gene tcdC;        6. If both tested antibiotic resistances are present along with all the features of the characteristic genotypical pattern: diagnosis of a hypervirulent C. difficile ribotype 027 strain (C.d.-027-hv).        
One disadvantage of previous laboratory practice which is of similar complexity for other variant and highly virulent C. difficile strains, lies primarily in the fact that it is very time-consuming: to isolate and culture the germ, to determine antibiotic resistance and for ribotyping, 7-10 days are required.